Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 716

Original
plasmid

Hybrid
plasmid

Restriction Enzyme
PstI

DNA ligase

+

R-plasmid

circularized
DNA

Restriction Enzyme
PstI

Foreign DNA

FIGURE 14.10 Insertion of a DNA gene into a plasmid.

divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally
for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells
will readily take up a plasmid. Because they are so small, they are easy to handle in a test
tube, and foreign genes can quite easily be incorporated into them using restriction enzymes
and DNA ligase.
One of the most common plasmids used is the R-plasmid (or pBR322). This plasmid
contains a replication origin, several recognition sequences for different restriction enzymes
(with names like PstI and EcoRI), and two marker genes, which confer resistance to different
antibiotics (ampicillin and tetracycline).
Fig. 14.10 shows how DNA fragments can be incorporated into a plasmid using restriction
and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of
one of the marker genes (we will see why this is useful later). The foreign DNA anneals with
the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words
a mixture or hybrid of bacterial and foreign DNA). Several other products are also formed:
some plasmids will simply reanneal with themselves to reform the original plasmid, and
some DNA fragments will join together to form chains or circles. These different products
cannot easily be separated, but it does not matter, as the marker genes can be used later to
identify the correct hybrid vector.

14.4.5.4. Gene Transfer
Vectors containing the genes we want must be incorporated into living cells so that they can
be replicated or expressed. The cells receiving the vector are called host cells, and once they
have successfully incorporated the vector, they are said to be transformed. Vectors are large
moleculeswhichdo not readily cross cell membranes, so themembranesmust bemade perme-
able in some way. There are different ways of doing this depending on the type of host cell.
1. Heat shockd Cells are incubated with the vector in a solution containing calcium ions at
0 C. The temperature is then suddenly raised to about 40 C. This heat shock causes some
of the cells to take up the vector, though no one knows why. This works well for bacterial
and animal cells.